The present invention relates to new polymers as reagents for the qualitative and quantitative measurement of ligands and receptors in physiological fluids.
In the past several years much attention has focused on the need to provide economical and rapid means of detecting the presence or absence of biologically active substances in physiological fluids. For example, the treatment of various diseases with drugs having a narrow therapeutic dosage range requires a safe and effective means for accurately determining the amount of drug in the body so that a proper dosage schedule may be tailored to the individual patient.
Further, there is a growing need for an immunoassay system which can be performed by a patient at home. Such a system may be used for example to monitor drug levels, detect vitamin deficiencies, and as a reliable test for pregnancy and fertility.
There presently exist several immunochemical methods of detecting ligands and receptors. Radioimmunoassay is the most widely used method. This method employs a molecule which has been labeled with a radioactive isotope. By combining an antibody to the ligand (ligand is the compound to be determined) with a small amount of ligand which has been labeled with a radioactive isotope and separating ligand-bound-to-antibody from ligand which is unbound, the amount of ligand present in the original sample is determined from the amount of radioactive ligand remaining in the supernatant solution. U.S. Pat. No. 3,709,868 describes such a radioimmunoassay.
Several non-isotopic immunoassay methods have been proposed to eliminate the disadvantages associated with radioactive materials. For example, the radioactive label may be replaced with an enzyme (U.S. Pat. No. 3,654,090). By the addition of a ligand the distribution of the enzyme labeled component over the bound and free fraction is altered. The amount of soluble or insoluble enzyme labeled component can be determined by the specific enzyme activity, said amount being a measure of the ligand in the sample. In a preferred form of the invention the conjugated enzyme is detected by a color reaction, i.e. either a colored substrate or a colored end-product participates in the catalytic reaction which allows spectrophotmetric measurement.
Another enzyme immunoassay (U.S. Pat. No. 3,817,837) is marketed under the trademark EMIT. This is a homogeneous technique (the separation step is eliminated) such that antibody (or any other suitable receptor) when bound to the enzyme labeled ligand substantially inhibits enzymatic activity, providing for different catalytic efficiencies between the bound and unbound form of the enzyme.
The reaction of a substrate labeled ligand with an enzyme to produce a fluorescent product is described in U.S. Pat. No. 4,226,978. When antibody is bound to the substrate labeled ligand the enzyme is prevented from reacting with the substrate. The intensity of fluorescence is proportional to the amount of ligand present in the sample.
U.S. Pat. No. 4,166,105 and 4,169,137 describe antigen detecting reagents which are prepared by covalently linking fluorescent dye molecules to an appropriate antibody through a polymer possessing reactive functional groups.
Each of the foregoing prior art systems require the use of sophisticated instrumentation or complex procedures to measure the level of the biologically active substances. In contradistinction, the present invention provides immunoassay systems which may be used in the home relying on visual observation to determine the presence of biological substances. Furthermore, the present reagents are advantageous over prior art reagents in that they are highly sensitive permitting the detection of small amounts of ligands or receptors, more stable resulting in a longer shelf life, easier to prepare, and can be used for the detection of both high and low molecular weight substances.